Coryneform bacteria with altered glucokinase activity in the production of L-lysine

ABSTRACT

The invention relates to alleles of the glk gene from coryneform bacteria coding for glucokinases, and to processes for the production of L-lysine by fermentation using bacteria containing such alleles.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application represents U.S. national stage of internationalapplication PCT/EP02/12419, which had an international filing date ofNov. 7, 2002 and which was published in English under PCT Article 21(2)on Jul. 3, 2003. The international application claims priority to Germanapplication 101 62 730.0, filed on Dec. 20, 2001.

FIELD OF THE INVENTION

The invention provides alleles of the glk gene from coryneform bacteriacoding for variants of glucokinase, and processes for the production ofL-lysine by fermentation using bacteria containing such alleles.

PRIOR ART

The amino acid L-lysine is used in human medicine and in thepharmaceuticals industry, in the foodstuffs industry and, veryespecially, in the feeding of animals.

It is known that amino acids are produced by fermentation of strains ofcoryneform bacteria, especially Corynebacterium glutamicum. Because oftheir great importance, attempts are continuously being made to improvethe production processes. Improvements to the processes may concernmeasures relating to the fermentation, such as, for example, stirringand oxygen supply, or the composition of the nutrient media, such as,for example, the sugar concentration during the fermentation, or workingup to the product form by, for example, ion-exchange chromatography, orthe intrinsic performance properties of the microorganism itself.

In order to improve the performance properties of such microorganisms,methods of mutagenesis, selection and mutant selection are employed.Such methods yield strains which are resistant to antimetabolites or areauxotrophic for metabolites that are important in terms of regulation,and which produce amino acids. A known antimetabolite is the lysineanalogue S-(2-aminoethyl)-L-cysteine (AEC).

For a number of years, methods of recombinant DNA technology have alsobeen used for improving the strain of L-amino acid-producing strains ofCorynebacterium, by amplifying individual amino acid biosynthesis genesand studying the effect on amino acid production.

The nucleotide sequence of the gene coding for the glucokinase ofCorynebacterium glutamicum can be found in patent application WO01/00844 under the Identification Code RXA02149 as Sequence No. 23.

The nucleotide sequence of the gene coding for the glucokinase ofCorynebacterium glutamicum can also be found in patent applicationEP-A-1108790 as Sequence No. 3484 and as Sequence No. 7066.

The nucleotide sequence has also been deposited in the data bank of theNational Center for Biotechnology Information (NCBI) of the NationalLibrary of Medicine (Bethesda, Md., USA) under Accession Number AX064897and under Accession Number AX123568.

The favorable action of the overexpression of the glk gene on lysineproduction is shown in EP-A-1106694.

OBJECT OF THE INVENTION

The inventors have set themselves the object of providing novel measuresfor the improved production of L-lysine by fermentation.

SUMMARY OF THE INVENTION

Where L-lysine or lysine is mentioned hereinbelow, it is to beunderstood as meaning not only the bases but also the salts, such as,for example, lysine monohydrochloride or lysine sulfate.

The invention provides replicable nucleotide sequences (DNA) originatingfrom coryneform bacteria, especially Corynebacterium glutamicum, andcoding for the enzyme glucokinase, wherein the associated amino acidsequences in SEQ ID No. 2 contain at position 213 any proteinogenicamino acid, with the exception of L-alanine.

Proteinogenic amino acids are understood as being the amino acids whichoccur in naturally occurring proteins, that is to say in proteins ofmicroorganisms, plants, animals and humans. They include the amino acidsL-glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine,L-threonine, L-cysteine, L-methionine, L-proline, L-phenylalanine,L-tyrosine, L-tryptophan, L-asparagine, L-glutamine, L-aspartic acid,L-glutamic acid, L-arginine, L-lysine, L-histidine and L-selenocysteine.

The invention also provides a replicable nucleotide sequence (DNA)originating from coryneform bacteria, especially Corynebacteriumglutamicum, and coding for the enzyme glucokinase, wherein theassociated amino acid sequence contains L-valine at position 213, shownin SEQ ID No. 4.

The invention also provides a replicable nucleotide sequence (DNA)originating from coryneform bacteria, especially Corynebacteriumglutamicum, and coding for the enzyme glucokinase, the base sequence ofwhich contains thymine at position 638, shown in SEQ ID No. 3.

The invention also provides plasmids (vectors) which contain thenucleotide sequences according to the invention and optionally replicatein coryneform bacteria.

The invention also provides coryneform bacteria which contain thenucleotide sequences according to the invention and in which thenucleotide sequences coding for glucokinase are optionally inoverexpressed form, wherein the associated amino acid sequences containa different proteinogenic amino acid at position 213 of SEQ ID No. 2.

Overexpression is understood as meaning an increase in the intracellularconcentration or activity of the glucokinases according to theinvention.

DETAILED DESCRIPTION OF THE INVENTION

By the measures of overexpression, the activity or concentration of thecorresponding protein is generally increased by at least 10%, 25%, 50%,75%, 100%, 150%, 200%, 300%, 400% or 500%, at the maximum up to 1000% or2000%, based on the activity or concentration of the protein in thestarting microorganism.

In order to achieve overexpression, the copy number of the correspondinggenes can be increased, or the promoter and regulation region or theribosome binding site, which is located upstream of the structural gene,can be mutated. Expression cassettes inserted upstream of the structuralgene have the same effect. By means of inducible promoters it isadditionally possible to increase the expression in the course of theproduction of L-lysine by fermentation. Expression is also improved bymeasures to prolong the life of the m-RNA. Furthermore, the enzymeactivity is also enhanced by preventing degradation of the enzymeprotein. The genes or gene constructs may either be present in plasmidswith a different copy number or be integrated and amplified in thechromosome. Alternatively, overexpression of the genes in question mayalso be achieved by changing the composition of the medium and themanner in which culturing is carried out.

For increasing the copy number of the glk alleles according to theinvention, plasmids which are replicated in coryneform bacteria aresuitable. Many known plasmid vectors, such as, for example, pZ1 (Menkelet al., Applied and Environmental Microbiology (1989) 64: 549-554),pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen etal., Gene 107:69-74 (1991)), are based on the cryptic plasmids pHM1519,pBL1 or pGA1. Other plasmid vectors, such as, for example, those whichare based on pCG4 (U.S. Pat. No. 4,489,160) or pNG2 (Serwold-Davis etal., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (U.S. Pat.No. 5,158,891), can be used in the same manner.

For increasing the copy number it is also possible to use the method ofchromosomal gene amplification, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for the duplication or amplification of the hom-thrB operon. Inthat method, the complete gene or allele is cloned into a plasmid vectorthat is able to replicate in a host (typically E. coli), but not in C.glutamicum. Suitable vectors are, for example, pSUP301 (Simon et al.,Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al.,Gene 145, 69-73 (1994)), PGEM-T (Promega Corporation, Madison, Wis.,USA), pCR2.1-TOPO (Shuman, Journal of Biological Chemistry 269:32678-84(1994); U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen, Groningen,Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541(1993)), pEM1 (Schrumpf et al., Journal of Bacteriology 173:4510-4516(1991)) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986)). The plasmidvector containing the gene or allele to be amplified is then transferredto the desired strain of C. glutamicum by conjugation or transformation.The method of conjugation is described, for example, in Schafer et al.(Applied and Environmental Microbiology 60, 756-759 (1994)). Methods oftransformation are described, for example, in Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a “cross-over” event, the resulting straincontains at least two copies of the gene or allele in question.

The invention provides replicable, especially endogenous, nucleotidesequences (DNA) originating from coryneform bacteria and coding for theenzyme glucokinase, wherein in the associated amino acid sequencesL-alanine at position 213 of SEQ ID No. 2 is replaced by a differentproteinogenic amino acid, especially L-valine, shown in SEQ ID No. 4.The invention also provides replicable, preferably endogenous,nucleotide sequences (DNA) originating from coryneform bacteria andcoding for the enzyme glucokinase, the associated base sequence of whichcontains thymine at position 638, shown in SEQ ID No. 3.

“Endogenous genes” or “endogenous nucleotide sequences” are understoodas being the genes or nucleotide sequences present in the population ofa species.

The invention relates also to vectors (plasmids) which contain thementioned nucleotide sequences and optionally replicate in coryneformbacteria.

Also claimed are coryneform bacteria in which the mentioned nucleotidesequences coding for enzyme glucokinase, are preferably in overexpressedform.

The invention provides a process for the production of L-lysine or offeed additives containing L-lysine, in which the following steps aregenerally carried out:

-   -   a) fermentation of coryneform bacteria containing endogenous        nucleotide sequences coding for the enzyme glucokinase, wherein        in the associated amino acid sequences L-alanine at position 213        has been replaced by a different proteinogenic amino acid,        preferably L-valine.        -   The alleles of the endogenous glucokinase gene are.            overexpressed under conditions suitable for the formation of            the enzyme glucokinase.    -   b) concentration of the L-lysine in the fermentation liquor,    -   c) isolation of the L-lysine or of the feed additive containing        L-lysine from the fermentation liquor, optionally    -   d) with constituents of the fermentation liquor and/or the        biomass (>0 to 100%).

Proteinogenic amino acids are to be understood as being all amino acidsthat are constituents of proteins or polypeptides. They are especially:L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid,L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine,L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,L-lysine, L-tryptophan, L-proline and L-arginine.

The wild form of the glk gene is contained in wild-type strains ofcoryneform bacteria, especially of the genus Corynebacterium. It isshown in SEQ ID No. 1. The wild-type protein is shown in SEQ ID No. 2.

Of the genus Corynebacterium, special mention is to be made of thespecies Corynebacterium glutamicum, which is known in the specialistfield. Known wild-type strains of the species Corynebacterium glutamicumare, for example,

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium melassecola ATCC17965    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020.

Strains having the designation “ATCC” can be obtained from the AmericanType Culture Collection (Manassas, Va., USA). Strains having thedesignation “FERM” can be obtained from the National Institute ofAdvanced Industrial Science and Technology (AIST Tsukuba Central 6,1-1-1 Higashi, Tsukuba Ibaraki, Japan). The mentioned strain ofCorynebacterium thermoaminogenes (FERM BP-153.9) is described in U.S.Pat. No. 5,250,434.

For the production of the glk alleles according to the invention whichcode for variants of glucokinase, characterized by an amino acidreplacement at position 213 of SEQ ID No. 2, methods of mutagenesisdescribed in the prior art are used.

It is possible to use for the mutagenesis conventional in vivo processesof mutagenesis using mutagenic substances such as, for example,N-methyl-N′-nitro-N-nitrosoguanidine or ultraviolet light.

It is also possible to use for the mutagenesis in vitro methods, suchas, for example, treatment with hydroxylamine (Miller, J. H.: A ShortCourse in Bacterial Genetics. A Laboratory Manual and Handbook forEscherichia coli and Related Bacteria, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1992) or mutagenic oligonucleotides (T. A.Brown: Gentechnologie für Einsteiger, Spektrum Akademischer Verlag,Heidelberg, 1993) or the polymerase chain reaction (PCR), as isdescribed in the handbook of Newton and Graham (PCR, SpektrumAkademischer Verlag, Heidelberg, 1994).

Further instructions for the production of mutations can be found in theprior art and in known textbooks of genetics and molecular biology, suchas, for example, the textbook of Knippers (“Molekulare Genetik”, 6thedition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that ofWinnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim, Germany,1990) or that of Hagemann (“Allgemeine Genetik”, Gustav Fischer Verlag,Stuttgart, 1986).

When in vitro methods are used, the glk gene described in the prior artis amplified by means of the polymerase chain reaction starting fromisolated total DNA of a wild-type strain and is optionally cloned intosuitable plasmid vectors, and the DNA is subsequently subjected to themutagenesis process. The person skilled in the art will findinstructions for the amplification of DNA sequences by means of thepolymerase chain reaction (PCR) inter alia in the handbook of Gait:Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK,1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag,Heidelberg, Germany, 1994). Suitable glk alleles are subsequentlyselected and studied using the above-described processes.

The invention provides a novel glk allele coding for a variant ofglucokinase, which allele is shown in SEQ ID No. 3.

The glk alleles according to the invention can be transferred intosuitable strains by the method of gene replacement, as is described inSchwarzer and Pühler (Bio/Technology 9, 84-87 (1991)) or Peters-Wendischet al. (Microbiology 144, 915-927 (1998)). In that method, theappropriate glk allele is cloned into a vector that is not replicativefor C. glutamicum, such as, for example, pK18mobsacB or pK19mobsacB(Jäger et al., Journal of Bacteriology 174: 5462-65 (1992)) or pCR®Blunt(Invitrogen, Groningen, Netherlands; Bernard et al., Journal ofMolecular Biology, 234: 534-541 (1993)) and the vector is thentransferred into the desired host of C. glutamicum by transformation orconjugation. After homologous recombination by means of a first“cross-over” event effecting integration and a suitable second“cross-over” event effecting an excision in the target gene or in thetarget sequence, incorporation of the mutation is achieved.

It may additionally be advantageous for the production of L-amino acids,in addition to using the glk alleles according to the invention, at thesame time to enhance, especially overexpress, one or more enzymes of thebiosynthesis pathway in question, of glycolysis, of the anapleroticpathway, of the citric acid cycle, of the pentose phosphate cycle, ofamino acid export and, optionally, regulatory proteins. The use ofendogenous genes is generally preferred.

“Endogenous genes” or “endogenous nucleotide sequences” are understoodas being the genes or nucleotide sequences and alleles present in thepopulation of a species.

The term “enhancement” in this context describes the increase of theintracellular activity of one or more enzymes (proteins) in amicroorganism that are coded for by the corresponding DNA, by, forexample, increasing the copy number of the gene or genes, using a strongpromoter or using a gene or allele that codes for a corresponding enzyme(protein) having a high level of activity, and optionally by combiningthose measures.

By the measures of enhancement, especially overexpression, the activityor concentration of the corresponding protein is generally increased byat least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, atthe maximum up to 1000% or 2000%, based on that of the wild-type proteinor on the activity or concentration of the protein in the startingmicroorganism.

Accordingly, for the production of L-lysine, in addition to using thevariant of the glk gene, it is also possible at the same time toenhance, especially overexpress, one or more genes selected from thegroup.

-   -   the gene dapA coding for dihydrodipicolinate synthase (EP-B 0        197 335),    -   the gene gap coding for glyceraldehyde-3-phosphate dehydrogenase        (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),    -   the gene eno coding for enolase (EP-A-1090998),    -   the gene tpi coding for triose phosphate isomerase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086),    -   the gene pgk coding for 3-phosphoglycerate kinase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086),    -   the gene zwf coding for glucose-6-phosphate dehydrogenase        (JP-A-09224661, EP-A-1108790, WO 01/70995, WO 01/98472, WO        01/04322),    -   the gene pyc coding for pyruvate carboxylase (DE-A-198 31 609,        EP-A-1108790),    -   the gene mqo coding for malate quinone oxidoreductase (Molenaar        et al., European Journal of Biochemistry 254, 395-403 (1998),        EP-A-1038969),    -   the gene lysC coding for a feed-back resistant aspartate kinase        (Accession No. P26512; EP-B-0387527; EP-A-0699759; WO 00/63388),    -   the gene lysE coding for the lysine export protein (DE-A-195 48        222, Vrljic et al., Molecular Microbiology 22(5), 815-826        (1996)),    -   the gene zwa1 coding for the Zwa1 protein (EP-A-1111062),    -   the gene gnd coding for 6-phosphogluconate dehydrogenase (WO        01/71012),    -   the gene opcA coding for a sub-unit of glucose-6-phosphate        dehydrogenase (Sequence No. 79 from WO 01/00844; WO 01/04322)

The enhancement of 6-phosphogluconate dehydrogenase can also beachieved, inter alia, by amino acid replacements, such as, for example,by the replacement of L-proline by L-serine, L-leucine, L-isoleucine orL-threonine at position 158 of the enzyme protein, and/or by thereplacement of L-serine by L-phenylalanine or L-tyrosine at position 361of the enzyme protein.

The enhancement of the sub-unit of glucose-6-phosphate dehydrogenase,for which the gene opcA codes, can also be achieved, inter alia, byamino acid replacements, such as, for example, by the replacement ofL-serine by L-phenylalanine or L-tyrosine at position 312 of the enzymeprotein.

It may also be advantageous for the production of L-lysine, in additionto using the alleles of the glk gene according to the invention, at thesame time to attenuate, especially diminish the expression of, one ormore endogenous genes selected from the group

-   -   the gene pck coding for phosphoenol pyruvate carboxykinase        (EP-A-1094111),    -   the gene pgi coding for glucose-6-phosphate isomerase        (EP-A-1087015, WO 01/07626, EP-A-1108790),    -   the gene poxB coding for pyruvate oxidase (EP-A-1096013),    -   the gene zwa2 coding for the Zwa2 protein (EP-A-1106693),    -   the gene fda coding for fructose-1,6-bisphosphate aldolase        (Accession No. X17313; von der Osten et al., Molecular        Microbiology 3 (11), 1625-1637 (1989)),    -   the gene hom coding for homoserine dehydrogenase (EP-A-0131171),    -   the gene thrB coding for homoserine kinase (Peoples, O. W., et        al., Molecular Microbiology 2 (1988): 63-72) and    -   the gene pfkb coding for phosphofructokinase (Sequence No. 57        from WO 01/00844).

The term “attenuation” in this context describes the diminution orexclusion of the intracellular activity of one or more enzymes(proteins) in a microorganism that are coded for by the correspondingDNA, by, for example, using a weak promoter or using a gene or allelethat codes for a corresponding enzyme having a low level of activity, orby inactivating the corresponding gene or enzyme (protein), andoptionally by combining those measures.

By the measures of attenuation, the activity or concentration of thecorresponding protein is generally lowered to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein, or of the activity or concentration of the protein inthe starting microorganism.

The attenuation of phosphofructokinase can also be achieved, inter alia,by amino acid replacements, such as, for example, by the replacement ofL-leucine by L-alanine, L-glycine or L-proline at position 109 of theenzyme protein.

The microorganisms produced according to the invention also form part ofthe invention and can be cultivated continuously or discontinuously bythe batch process or by the fed batch or repeated fed batch process forthe purposes of the production of L-amino acids. A summary of knowncultivation methods is described in the textbook of Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas(Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of the strainsin question in a suitable manner. Descriptions of culture media forvarious microorganisms are to be found in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

There may be used as the carbon source sugars and carbohydrates, suchas, for example, glucose, saccharose, lactose, fructose, maltose,molasses, starch and cellulose, oils and fats, such as, for example,soybean oil, sunflower oil, groundnut oil and coconut oil, fatty acids,such as, for example, palmitic acid, stearic acid and linoleic acid,alcohols, such as, for example, glycerol and ethanol, and organic acids,such as, for example, acetic acid. Those substances may be usedindividually or in the form of a mixture.

There may be used as the nitrogen source organic nitrogen-containingcompounds, such as peptones, yeast extract, meat extract, malt extract,corn steep liquor, soybean flour and urea, or inorganic compounds, suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources may be usedindividually or in the form of a mixture.

There may be used as the phosphorus source phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts. The culture medium must alsocontain salts of metals, such as, for example, magnesium sulfate or ironsulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, may be used in addition tothe above-mentioned substances. Suitable precursors may also be added tothe culture medium. The mentioned substances may be added to the culturein the form of a single batch, or they may be suitably fed in during thecultivation.

In order to control the pH of the culture, basic compounds, such assodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidcompounds, such as phosphoric acid or sulfuric acid, are expedientlyused. In order to control the development of foam, anti-foams, such as,for example, fatty acid polyglycol esters, may be used. In order tomaintain the stability of plasmids, suitable substances having aselective action, such as, for example, antibiotics, may be added to themedium. In order to maintain aerobic conditions, oxygen or gas mixturescontaining oxygen, such as, for example, air, are introduced into theculture. The temperature of the culture is normally from 20° C. to 45°C. and preferably from 25° C. to 40° C. The culture is continued untilthe maximum amount of the desired product has formed. That aim isnormally achieved within a period of from 10 hours to 160 hours.

Methods of determining L-amino acids are known from the prior art. Theanalysis may be carried out, for example, as described in Spackman etal. (Analytical Chemistry, 30, (1958), 1190) by anion-exchangechromatography with subsequent ninhydrin derivation, or it may becarried out by reversed phase HPLC, as described in Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

The process according to the invention is used for the production ofL-lysine by fermentation.

The concentration of L-lysine can optionally be adjusted to the desiredvalue by the addition of L-lysine.

The present invention is explained in greater detail below by means ofembodiment examples.

EXAMPLE 1

Amplification and sequencing of the DNA of the glk allele of strainDM1454

The Corynebacterium glutamicum strain DM1454 was prepared from C.glutamicum ATCC13032 by repeated, undirected mutagenesis, selection andmutant selection. The strain is resistant to the lysine analogueS-(2-aminoethyl)-L-cysteine.

Chromosomal DNA is isolated from strain DM1454 by the conventionalmethods (Eikmanns et al., Microbiology 140: 1817-1828 (1994)). By meansof the polymerase chain reaction, a DNA section carrying the glk gene orallele is amplified. On the basis of the sequence of the glk gene knownfor C. glutamicum (Sequence No. 3484 and Sequence No. 7066 fromEP-A-1108790), the following primer oligonucleotides are selected forthe PCR:

(SEQ ID No. 6) glk_XL-A1: 5′ ga tct aga-gct tct cga cga tcc gat cc 3′(SEQ ID No. 7) glk_XL-A2: 5′ ga tct aga-cat tat ctg cgg tgc ggt cc 3′

The primers shown are synthesized by MWG Biotech (Ebersberg, Germany),and the PCR reaction is carried out according to the standard PCR methodof Innis et al. (PCR protocols. A Guide to Methods and Applications,1990, Academic Press). The primers allow the amplification of a DNAsection having a length of approximately 1.65 kb and carrying the glkgene or allele. The primers additionally contain the sequence for acleavage site of the restriction endonuclease xbaI, which is marked inthe nucleotide sequence shown above by underlining.

The amplified DNA fragment having a length of approximately 1.65 kb,which carries the glk allele of strain DM1454, is identified byelectrophoresis in a 0.8% agarose gel, isolated from the gel andpurified by conventional methods (QIAquick Gel Extraction Kit, Qiagen,Hilden).

The nucleotide sequence of the amplified DNA fragment, or PCR product,is determined by MWG Biotech (Ebersberg, Germany) by sequencing. Thesequence of the PCR product is shown in SEQ ID No. 5. The sequence ofthe coding region is additionally shown in SEQ ID No. 3. The amino acidsequence of the associated glucokinase protein, determined by means ofthe Patentin program, is shown in SEQ ID No. 4.

At position 638 of the nucleotide sequence of the coding region of theglk allele of strain DM1454 there is the base thymine (SEQ ID No. 3). Atthe corresponding position of the wild-type gene there is the basecytosine (SEQ ID No. 1).

At position 213 of the amino acid sequence of the glucokinase protein ofstrain DM1454 there is the amino acid valine (SEQ ID No. 4). At thecorresponding position of the wild-type protein there is the amino acidalanine (SEQ ID No. 2).

The glk allele, which contains the base thymine at position 638 of thecoding region and accordingly codes for a glucokinase protein whichcontains the amino acid valine at position 213 of the amino acidsequence, is referred to hereinbelow as the glk_A213V allele. In thedesignation “glk_A213V”, A represents alanine, V represents L-valine and213 indicates the position of the amino acid replacement (see SEQ ID No.2 and 4).

EXAMPLE 2

Replacement of the glk wild-type gene of strain DSM5715 by the glk_A213Vallele

2.1 Construction of the Replacement Vector pK18mobsacB_glk_A213V

The DNA fragment approximately 1.65 kb in length which is described inExample 1 and was prepared by means of PCR and which carries theglk_A213V allele is inserted into the chromosome of C. glutamicum strainDS15715 by means of replacement mutagenesis with the aid of the sacBsystem described in Schafer et al. (Gene, 14, 69-73 (1994)). This systemallows the preparation or selection of allele replacements which takeplace by homologous recombination. Strain DSM5715 is aleucine-requiring, aminoethylcysteine-resistant and L-lysine-producingmutant of Corynebacterium glutamicum ATCC13032. The strain is describedin EP-A-0435 132.

To that end, the glk_A213V fragment approximately 1.65 kb in size iscleaved with restriction endonuclease XbaI, identified byelectrophoresis in a 0.8% agarose gel, and then isolated from the geland purified by conventional methods (QIAquick Gel Extraction Kit,Qiagen, Hilden).

The mobilizable cloning vector pK18mobsacB is digested with restrictionenzyme XbaI and the ends are dephosphorylated with alkaline phosphatase(Alkaline Phosphatase, Boehringer Mannheim, Germany). The vector soprepared is mixed with the approximately 1.6 kb glk_A213V fragment andthe batch is treated with T4-DNA ligase (Amersham-Pharmacia, Freiburg,Germany).

E. coli strain S17-1 (Simon et al., Bio/Technology 1: 784-791, 1993) isthen transformed with the ligation batch (Hanahan, In. DNA cloning Apractical approach. Vol.1. ILR-Press, Cold Spring Harbor, N.Y., 1989).Selection of the plasmid-carrying cells is carried out by plating outthe transformation batch on LB agar (Sambrook et al., Molecular Cloning:A Laboratory Manual. 2^(nd) Ed. Cold Spring Harbor, N.Y., 1989)supplemented with 25 mg/l kanamycin.

Plasmid DNA is isolated from a transformant by means of the QIAprep SpinMiniprep Kit from Qiagen and is checked by restriction cleavage with theenzyme BamHI and subsequent agarose gel electrophoresis. The plasmid isnamed pK18mobsacB_glk_A213V and is shown in FIG. 1.

2.2 Allele Replacement

The vector pK18mobsacB_glk_A213V mentioned in Example 2.1 is transferredinto C. glutamicum strain DSM5715 by conjugation according to a protocolof Schafer et al. (Journal of Microbiology 172: 1663-1666.(1990)). Thevector cannot replicate independently in DSM5715 and is only retained inthe cell if it has been integrated into the chromosome as the result ofa recombination event. The selection of transconjugants, i.e. of cloneshaving integrated pK18mobsacB_glk_A213V, is carried out by plating outthe conjugation batch on LB agar (Sambrook et al., Molecular Cloning: ALaboratory Manual. 2^(nd) Ed. Cold Spring Harbor, N.Y., 1989)supplemented with 15 mg/l kanamycin and 50 mg/l nalidixic acid.Kanamycin-resistant transconjugants are spread onto LB agar plates with25 mg/l kanamycin and incubated for 24 hours at 33° C. For the selectionof mutants in which the excision of the plasmid has taken place as aresult of a second recombination event, the clones are cultivatednon-selectively in LB liquid medium for 30 hours, then spread onto LBagar with 10% sucrose and incubated for 16 hours.

Plasmid pK18mobsacB_glk_A213V, like the starting plasmid pK18mobsacB,contains, in addition to the kanamycin resistance gene, a copy of thesacB gene coding for levan sucrase from Bacillus subtilis. Thesucrose-inducible expression leads to the formation of levan sucrase,which catalyses the synthesis of the product levan, which is toxic forC. glutamicum. Therefore, only those clones in which the integratedpK18mobsacB_glk_A213V has excised as a result of a second recombinationevent grow on LB agar with sucrose. In dependence on the position of thesecond recombination occurrence in relation to the site of mutation, theallele replacement or the incorporation of the mutation takes place atthe excision, or the original copy remains in the chromosome of thehost.

Approximately 40 to 50 colonies are tested for the phenotype “growth inthe presence of sucrose” and “non-growth in the presence of kanamycin”.In 4 colonies exhibiting the phenotype “growth in the presence ofsucrose” and “non-growth in the presence of kanamycin”, a region of theglk gene, starting from the sequencing primer gl1 (SEQ ID No. 8),spanning the A213V mutation is sequenced by GATC Biotech AG (Constance,Germany) in order to demonstrate that the mutation of the glk_A213Vallele is present in the chromosome. The primer gl1 used therefor issynthesized by GATC Biotech AG:

(SEQ ID No. 8) gll: 5′ gga aca tga tgc caa ctc ag 3′

In that manner a clone is identified that contains the base thymine atposition 638 of the coding region of the glk gene and accordinglypossesses the glk_A213V allele. That clone is referred to as strainDSM5715glk_A213V.

EXAMPLE 3

Production of Lysine

The C. glutamicum strain DSM5715glk_A213V obtained in Example 2 iscultivated in a nutrient medium suitable for the production of lysine,and the lysine content in the culture supernatant is determined.

To that end, the strain is first incubated on agar plate for 24 hours at33° C. Starting from that agar plate culture, a preliminary culture isinoculated (10 ml of medium in a 100 ml Erlenmeyer flask). MM medium isused as the medium for the preliminary culture. The preliminary cultureis incubated for 24 hours at 33° C. at 240 rpm on a shaker. From thatpreliminary culture, a main culture is inoculated, so that the initialOD (660 nm) of the main culture is 0.1 OD. Mm medium is also used forthe main culture.

MM medium CSL 5 g/l MOPS 20 g/l Glucose (autoclaved separately) 50 g/lSalts: (NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(sterilized by filtration) 0.3 mg/l Thiamin * HCl (sterilized by 0.2mg/l filtration) L-leucine (sterilized by 0.1 g/l filtration) CaCO₃ 25g/l CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) andthe salt solution are adjusted to pH 7 with ammonia water andautoclaved. The sterile substrate and vitamin solutions and the dryautoclaved CaCO₃ are then added.

Cultivation takes place in a volume of 10 ml in a 100 ml Erlenmeyerflask with baffles. Cultivation takes place at 33° C. and 80% humidity.

After 72 hours, the OD is determined at a measuring wavelength of 660 nmusing a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount oflysine formed is determined by means of an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion-exchange chromatographyand post-column derivation with ninhydrin detection.

The result of the test is shown in Table 1.

TABLE 1 OD Lysine HCl Strain (660 nm) g/l DSM5715 8.2 13.57DSM5715glk_A213V 8.8 15.22

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1: Map of plasmid pK18mobsacB_glk_A213V.

The abbreviations and names used have the following meanings. Wherenumbers of base pairs are given, they are approximate values which areobtained within the scope of the reproducibility of measurements.

-   -   Kan: kanamycin resistance gene    -   BamHI: cleavage site of the restriction enzyme BamHI    -   XbaI: cleavage site of the restriction enzyme XbaI    -   glk: glk_A213V allele    -   sacB: sacB gene    -   RP4-mob: mob region having the origin of replication for the        transfer (oriT)    -   oriV: origin of replication V

1. An isolated protein comprising the amino acid sequence of SEQ IDNO:2, wherein: a) the amino acid at position 213 is an L-amino acidother than L-alanine and b) said protein has glucokinase activity. 2.The isolated protein of claim 1, wherein said amino acid at position 213is L-valine.
 3. The isolated protein of claim 1, wherein said amino acidsequence consists of the amino acid sequence of SEQ ID NO:2 but whereinthe amino acid at position 213 is an b-amino acid other than L-alanine.4. The isolated protein of claim 3, wherein said amino acid at position213 is L-valine.
 5. An isolated nucleic acid comprising a nucleotidesequence encoding the protein of any one of claims 1-4.
 6. An isolatednucleic acid encoding the protein of claim 4 and having glucokinaseactivity, said isolated nucleic acid comprising the nucleotide sequenceof SEQ ID NO:3.
 7. A vector comprising the nucleotide sequence of thenucleic acid of claim
 5. 8. A coryneform bacteria transformed with thevector of claim
 7. 9. A process for the production of L-lysine or of afeed additive containing L-lysine, comprising: a) fermenting thecoryneform bacteria of claim 8 under conditions suitable for theproduction of L-lysine, and b) isolating said L-lysine or thefermentation liquor containing said L-lysine.
 10. The process of claim 9wherein said coryneform bacteria are of the species Coryneformglutamicum.
 11. A process for the production of a composition ofL-lysine or of a feed additive containing L-lysine, comprising: a)fermenting coryneform bacteria containing a nucleotide sequence encodingthe enzyme glucokinase, wherein said glucokinase comprises the aminoacid sequence of SEQ ID NO:2 and in the encoded amino acid sequence,L-alanine at position 213 has been replaced by a different proteinogenicamino acid, b) concentrating the L-lysine in the fermentation liquor, c)isolating said L-lysine or the fermentation liquor containing saidL-lysine.
 12. The process of claim 11, wherein said amino acid atposition 213 is L-valine.
 13. The process of either claim 11 or claim12, wherein said composition of L-lysine or said fermentation liquorcomprises >0 to 100% of the constituents of said fermentation liquorand/or of the biomass present during fermentation.
 14. An isolatednucleic acid consisting of a nucleotide sequence encoding the protein ofany one of claims 1-4.
 15. An isolated nucleic acid, encoding theprotein of claim 4, wherein said nucleic acid consists of the nucleotidesequence of SEQ ID NO:3.